Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes.
A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.
Ninfali
P
P
Institute of Biological Chemistry G. Forniani, University of Urbino, Italy.
Orsenigo
I
I
Baronciani
L
L
Rapa
S
S
D000085:Acetates; D000818:Animals; D002845:Chromatography; D002951:Citrates; D019343:Citric Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D009249:NADP; D011817:Rabbits; D013696:Temperature
D000085:Acetates; D002951:Citrates; D019343:Citric Acid; D009249:NADP; D005954:Glucosephosphate Dehydrogenase